The separation of DNA fragments by polyacrylamide or agarose gel electrophoresis is a well-established and widely used tool in molecular biology (Sharp, P.A. et al., xe2x80x9cDetection of two restriction endonucleases activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis,xe2x80x9d (1973) Biochemistry 12:3055). The standard technique for viewing the positions of the separated fragments in a gel involves the use of an ultra-violet (UV) transilluminator (Brunk, C. F. and Simpson, L., xe2x80x9cComparison of various ultraviolet sources for fluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels,xe2x80x9d (1977) Analytical Biochemistry 82:455). This procedure involves first staining the gel with a fluorescent dye such as ethidium bromide or SYBR(copyright) Green I. The DNA fragments, which bind the dye, are then visualized by placing the gel on a light-box equipped with a UV light-source. Typically the UV source, in combination with a built-in filter, provides light with an excitation maximum of around 254, 300 or 360 nm. The UV light causes the DNA-bound dye to fluoresce in the red (ethidium bromide) or green (SYBR Green I) regions of the visible light spectrum. The colored fluorescence allows visualization and localization of the DNA fragments in the gel. The visualization of DNA in a gel is used either to assess the success of a gene cloning reaction as judged by the size and number of DNA fragments present, or to identify a particular sized fragment which can be cut out from the gel and used in further reaction steps.
Transilluminators used in the art to visualize fluorophors are described in a number of patents, including U.S. Pat. Nos. 5,347,342, 5,387,801, 5,327,195, 4,657,655, and 4,071,883. Clinical examination of skin anomalies causing fluorescence have been described in U.S. Pat. No. 5,363,854 using visible light images as a control.
The use of UV light for viewing molecules in gels has two major disadvantages: (1) It is dangerous. The eyes are very sensitive to UV light and it is an absolute necessity that the viewer wear eye-protection, even for brief viewing periods, to prevent the possibility of serious damage. More prolonged exposure to UV light results in damage to the skin tissues (sunburn) and care must be taken to minimize skin exposure by wearing gloves, long-sleeved jackets and a full-face mask. (2) DNA samples are damaged by exposure to UV light. It has recently been documented by Epicentre Technologies that a 10-20 second exposure to 305 nm UV light on a transilluminator is sufficient to cause extensive damage to the DNA. This period of time is the absolute minimum required to excise a DNA band from a gel.
An alternative to UV transillumination involves the use of laser light sources. However, the use of laser light is not applicable to the simple and direct viewing of a DNA gel by the human eye. The extremely small cross-section of the laser light beam requires that a typical DNA gel be scanned by the laser, the fluorescence intensity at each point measured electronically and stored digitally before a composite picture of the DNA gel is assembled for viewing using computer software.
Visible light boxes for artists"" uses are known to the art for visualizing non-fluorescing materials, e.g., as described in U.S. Pat. No. 3,802,102. The use of visible light to detect certain fluorescent dyes is suggested, e.g., in Lightools Research web page. However, no enabling disclosure for making such devices is provided. None of these references provides devices or systems for viewing fluorescence patterns using visible light.
Despite the recent development of dyes fluorescing in the visible spectrum (Haugland, R. [1996] xe2x80x9cHandbook of Fluorescent Probes and Research Chemicals, Sixth Edition,xe2x80x9d Molecular Probes, Inc., Eugene, Oreg., pp. 13-18, 25-29, 29-35), transilluminators and other devices to take advantage of the properties of such dyes have not been made available to the public. It is an object of this invention to provide devices and methods for directly and indirectly viewing and measuring patterns of fluorescence not involving the use of UV transillumination but rather being capable of using sources of visible light such as ordinary lamps, as opposed to lasers and the focused lights used in standard fluorometers.
All publications referred to herein are incorporated by reference.
Avisible light system is provided for detection of patterns of fluorescence emitted by fluorophors capable of emitting light of an emitted wavelength range (emission spectrum) when excited by light of an excitation wavelength range (excitation spectrum). In one embodiment, the excitation wavelength range must be different from the emitted wavelength range, although these ranges may overlap, and at least a portion of the non-overlapping portion of the emitted wavelength range must be within the visible spectrum. Both the exciting and emitted wavelength ranges are within the visible spectrum.
In preferred embodiments, using color filters, light of the xe2x80x9cexcitation typexe2x80x9d for the fluorophor is light within the excitation wavelength range for the fluorophor, and light of the xe2x80x9cemitted typexe2x80x9d is light within the emitted wavelength range for the fluorophor. The first filter preferably transmits at least about 70% of the light from the light source in the excitation wavelength range, and the second filter transmits at least about 95% of the light in the emitted wavelength range. The term xe2x80x9cfilterxe2x80x9d as used herein includes combinations of filters.
In other embodiments using polarizing filters, the first filter transmits the light from the source in a narrow range of orientations, and the second filter is oriented to exclude light from the source, i.e., transmits only light orthogonal to that passed by the first filter, so that only light emitted by the fluorophor passes through the second filter.
This invention comprises a visible light system comprising:
a) a light source capable of producing visible light of the excitation type for the fluorophors;
b) a first optical filter placed between said light source and said fluorophors, which is capable of transmitting light from said light source of the excitation type for said fluorophors and of preventing transmission of at least a portion of the light from said light source of said emitted type; and
c) a second optical filter placed between said fluorophors and a light detector which second filter is capable of transmitting light of said emitted type and of preventing transmission of light from said light source of said excitation type, to form a viewable image of the pattern of fluorophors.
The fluorophors may be any fluorophors known or readily available to those skilled in the art, and are preferably used in the form of fluorophors bound to or in a biological sample. Fluorophors may be used to detect and quantify any desired substance to which they can be attached or into which they can be incorporated, e.g. organic molecules such as proteins, nucleic acids, carbohydrates, pigments, and dyes, inorganic molecules such as minerals, bacteria, eukaryotic cells, tissues and organisms. Fluorophors may also be an intrinsic part of an organism or substance to be detected, e.g., various dyes and pigments found in, for example, fungi, fish, bacteria and minerals.
The system of this invention may be incorporated into an integrated device such as a horizontal or vertical gel electrophoresis unit, scanner or other device in which detection of fluorescence is required.
The devices and methods of this invention are especially useful for viewing patterns, i.e., two-dimensional and three-dimensional spatial arrangements of fluorophors. Fluorescence detectors such as found in fluorometers are able to detect only the presence and intensity of fluorescence, and rather than generating an image generate a stream of data which must be interpreted by machine. The present invention allows direct viewing of two-dimensional (or three-dimensional) patterns of fluorophors by the human eye. Such patterns of fluorophors include the spatial arrangement of fluorophors on DNA on a gel, or of fluorophors on a TLC plate, the spatial distribution of fluorophors in test tubes in a rack, the spatial distribution of fluorophors in fungus or bacteria on skin, or on meat meant for human consumption, or the spatial arrangement of fluorescent fish in a tank. The images of patterns of fluorescence generated by the methods and devices of this invention may be viewed over time and may be photographed, digitized, stored and otherwise manipulated by machine but, in all cases, a two- or three-dimensional image is generated. The light source should not be a laser, and any mechanical detector used herein, like the human eye, preferably includes an array of photodetectors.
The light source should produce minimal light in the ultraviolet range, i.e., less than 1% of its light should be in the ultraviolet range, or the first filter should effectively screen out ultraviolet light, preferably to a level less than 1%, to prevent damage to DNA being viewed in the system. Even when using polarizing filters, a blue filter is preferably used as part of or in addition to the first filter to prevent DNA damage. Alternatively, the diffuser may be used to filter out residual UV light, and the diffuser and first filter can be combined into one sheet of material. (Most blue filters filter out ultraviolet light as well as visible light in wavelengths longer than blue.)
The light detector or xe2x80x9cviewerxe2x80x9d used to detect the fluorescence of the fluorophor using this system may be a viewer""s eye, or a device such as an optical scanner or charge coupled device camera for inputting a digitized image into a computer, or a camera. Such devices may also comprise means for quantifying the light within the emitted wavelength range reaching the viewer, and may also comprise means, such as a properly programmed computer as is known to the art, for converting such quantitative measurements to values for the amount of biological material present in the sample being measured.
The first filter is capable of filtering out light from the light source of the emitted type for the fluorophors. This means that at least some of the light from the light source of the emitted type is filtered out by the first filter. In many cases, the excitation and emission spectra for the fluorophors being used overlap. The first filter need only absorb light in a portion of the emission spectrum, usually the upper wavelength end thereof.
In some embodiments, the first filter may be an integral part of the support for the fluorophor or of the material or medium containing the fluorophor. For example, the first filter may serve as the gel support of a transilluminator device on which fluorophor-containing material in gel is placed. The gel itself, e.g., impregnated with pigment such as blue pigment, may serve as the first filter.
In some embodiments, as more fully described below, the second filter may be adapted to be placed over the human eye, e.g. as lenses for glasses to be worn by a human viewer, or may be adapted to be attached to the lens of an optical scanner or camera. The second filter may also serve as a safety lid for an electrophoresis unit or as a wall for the container for the fluorophor-containing material. The term xe2x80x9cattachedxe2x80x9d in this context means both removably attached or built in as an integral part of a device. Also in some embodiments described below, the light source may be a handheld light source held behind the sample or preferably in front of the sample and at an angle to the viewer. The handheld unit for holding the light source also preferably comprises the first optical filter as part of the casing.
The fluorophor-containing material may be transparent or opaque, and the system may be configured to allow light from the light source to pass directly through the first filter, the fluorophor-containing material, and the second filter to reach the viewer in the case of a transparent medium, or to allow light from the light source to pass through the first filter to strike the fluorophor-containing material, allowing emitted light to xe2x80x9cbouncexe2x80x9d back from the medium toward the viewer, first passing through the second filter. The configuration of optical components may occupy any angle from just over 0xc2x0 to 180xc2x0. The angle is that formed by lines drawn from the lamp to the sample and from the sample to the detector.
The term xe2x80x9ctransilluminatorxe2x80x9d as used herein means a device (other than a fluorometer requiring placement of fluorophor-labeled sample in a specially constructed sample holder) which allows light to shine through a surface in or on which a fluorophor-containing material has been placed, and includes horizontal electrophoresis devices and other devices in which fluorescent-containing materials are distributed on a surface.
Also provided are methods for making such systems and devices incorporating the light source and filters described above for viewing patterns of fluorescences emitted by fluorophors, said method comprising:
(a) providing a light source capable of producing light in the visible spectrum;
(b) placing said fluorophors spaced apart from said light source;
(c) placing a first optical filter between said light source and said fluorophors, said filter being capable of transmitting light from said light source of the excitation type for said fluorophors and of preventing transmission of light from said light source of the emitted type for said fluorophors; and
(d) placing a second optical filter between said fluorophors and a light detector, said second filter being capable of transmitting light of said emitted type and of preventing transmission of light from said light source of said excitation type.
Also provided are methods for viewing a pattern of fluorescence emitted by fluorophors capable of emitting light of an emitted type when excited by light of an excitation type different from said emitted type, at least a detectable portion of said emitted type being present in visible light, said method comprising:
(a) shining visible light on said fluorophors through a first optical filter which is capable of transmitting light of said excitation type and of preventing transmission of light of said emitted type, whereby said fluorophor emits light of said emitted type;
(b) passing light emitted by said fluorophor through a second optical filter which is capable of transmitting light of said emitted type and of absorbing light from the light source of said excitation type to form an image of said pattern of fluorescence; and
(c) viewing said image.
Devices of this invention use visible rather than ultraviolet light for exciting and viewing fluorescence. Preferred embodiments of this invention using light sources of around 9 W emit even less dangerous UV light than standard fluorescent tubes used in most offices and laboratories. Using visible light allows the integrity of DNA being viewed to be maintained. The devices of this invention allow detection of as little as 0.1-1 ng of DNA, equal to or slightly better than a 312 nm UV transilluminator. Using a charge-coupled device (CCD) camera, it is possible to detect levels as low as tens of picograms of SYBR Gold-stained DNA. Viewing may be done by eye or by an imaging device such as a camera or computer scanner using both conventional photography and digital imaging systems.